Trypan blue

A Trypan blue dye exclusion assay was conducted to assess the effects of Adina rubella Hance stem, branch, leaf, bark, heartwood extracts, and Picroside III on the proliferation of AML cells. The AML cells were treated with these extracts in 12-well plates. Trypan blue (Sigma, St Louis, MO, USA) staining was performed daily for four consecutive days. The concentration of Trypan blue used was 0.4%, and cells were stained by mixing an equal volume of 0.4% Trypan blue solution with the cell suspension at a 1:1 ratio. The stained cells were then analyzed after 1 min. The viable cell count was then determined using a hemocytometer.

The following primary (fluorescent)-labeled monoclonal antibodies directed against human antigens were used: FITC-labeled anti-EpCAM (clone VU-1D9, STEMCELL Technologies Germany GmbH, Köln, Germany, APC-labeled anti-HLA-B7 (clone BB7.1), BioLegend Europe B.V., Amsterdam, The Netherlands, APC-labeled anti-IL2 (clone MQI-17H12, and CaptureSelect™ Biotin Anti-IgG-CH1 conjugate (were from Thermo Fisher Scientific, Waltham, MA, USA). Anti-human IFNAR1 (anifrolumab) #SIM0022 was from BioXCell, Lebanon, NH, USA. The neutralizing IL2 antibody (clone 5334) was from R&D Systems, Inc., Minneapolis, NE, USA. The following reagents were used: trypan blue (Sigma Aldrich, Zwijndrecht, The Netherlands), Streptavidin-AlexaFluor™647 (Thermo Fisher Scientific, Waltham, MA USA), FITC-labeled Annexin-V (ImmunoTools GmbH, Friesoythe, Germany), and propidium iodide (PI) (Thermo Fisher Scientific, Waltham, MA USA). The following recombinant proteins were used: human IFNα-2b and human IL2. They were from ImmunoTools GmbH, Friesoythe, Germany. Biotinylated MHC I-Strep HLA-B*0702, CMV pp65 (TPRVTGGGAM), and APC-labeled strep-Tactin were from IBA Lifesciences GmbH, Göttingen, Germany.

ChemicalFetal bovine serum (FBS) was purchased from Gibco (F7524, USA). Methylene blue, MTT, trypan blue, DMEM, penicillin, trypsin-EDTA, streptomycin, dimethyl sulfoxide (DMSO), DPBF (1,3-Diphenylisobenzofuran), and surfactants (P80, SPA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of analytical grade.
InstrumentsThe CAP device employed in this study was purchased from a satellite-based company (Semnan, Iran). It features a copper tube as its central electrode and a copper surface ring as the second electrode. The former is connected to a high-voltage power supply, while the latter is connected to the ground. Helium gas was selected as the carrier gas due to its low breakdown voltage, which facilitates the production of homogeneous and uniform plasma. Specifically, helium gas with a gas flow rate of 4–5 ml/min was utilized to generate a plasma jet. The plasma jet length in the atmospheric ambient was approximately 20 mm.
Nano micelles synthesisMicelle-MB was prepared using a simple equilibrium method. During stirring, MB and two nonionic surfactants with two completely different hydrophilic–hydrophobic balances (HLBs) were added to the aqueous medium. Surfactants (SPA and P80) and MB were dissolved separately in water to create a uniform solution (a 7.5 mM stock of each surfactant was prepared independently). Then, different percentages of the two surfactants were combined. Following the ratio of two surfactants used in the MD simulation and a series of experimental iterations conducted within the laboratory, it was determined that 2/3 of the ultimate micelle composition consisted of SPA. In this way, a solution of 15 μM MB in micelles was prepared with SPA (HLB of 4.3) with 60% and P80 (HLB of 15) with 40% of the total portion, so an HLB of 8.58 was obtained. The final concentrations of SPA and P80 for the new compounds that were obtained were 2.5 mM and 1.5 mM, respectively. Nonetheless, the concentration for obtaining the polydispersity index (PDI) and the average size of the particles (Z average) was 1.5 mM (SPA) and 1 mM (P80). The final HLB was calculated using the following formula:
Final HLB= (SPA_{percentage in micelle} × HLB_SPA) + (P80_{percentage in micelle} × HLB_P80) (1)
= (0.6×4.3) + (0.4 ×15) = 8.58
Nano micelles characterizationUltraviolet-visible spectra were obtained using a spectrophotometer (UNICO 2100-UV, China). Also, Dynamic light scattering (DLS) measurements were performed using a nanoparticle analyzer (HORIBA SZ-100, Japan) equipped with a helium-neon (He-Ne) laser (633 nm).
Assessment of singlet oxygen production1,3-diphenylisobenzofuran (DPBF) obtained from Sigma-Aldrich (St. Louis, MO, USA) was used to perform singlet oxygen dosimetry. First, a 0.115mM solution of DPBF in ethanol was prepared. Second, to prepare the dosimeter, different proportions of DPBF and Micelle-MB were made by trial and error, and many aquatic environment tests were conducted since DPBF was destroyed in some high concentrations of micelles. Finally, a ratio of 2/3 DPBF in solution (Water + Micelle-MB + DPBF) was used. Then, the absorption spectrum was recorded at 0.076mM (0.115 mM × 2/3 ).
Cell cultureThe human glioblastoma U87-MG cell line was obtained from the Pasteur Institute of Iran. In DMEM medium supplemented with 10% FCS and 1% antibiotics, U87-MG cells were grown continuously as monolayers in 75-cm² plastic tissue culture flasks. All cellular cultures were maintained in an incubator with a humidified atmosphere at 37 °C and 5% CO₂.
MB and micelle-MB cytotoxicity assayThe U87-MG cell line was seeded in 96-well plates at 7000 cells/well density and incubated for 48 hr. Then, the cells were incubated with seven different concentrations of MB (0, 2.5, 5, 10, 15, 20, 40 mM) for 4 hr. The cells exposed to MB were subsequently placed in an incubator for 48 hr. Cell viability was assessed using the MTT assay with an ELISA reader (Stat Fax-2100 Awareness, Mountain View, CA, USA). The IC₅₀ for MB (concentration of MB which inhibits cell proliferation with 50%) was subsequently determined.
Experimental treatmentsThe U87-MG cell line was cultured in 96-well plates at a density of 7000 cells per well and incubated for 48 hr. Subsequently, the cells were exposed to a 15 µM concentration of MB during incubation. The cellular wells were subjected to the Plasma jet current (Current = 360-380 mA; the plasma jet tip’s distance to the plate’s bottom was 25 mm) for different durations (0, 30, 60, and 90 sec) at ambient temperature. The treated cells were then incubated at 37 °C for 48 hr.
Cell survival assayThe viability of U- 87 MG cells was determined using the MTT assay. To perform this assay, following the 48 hr incubation, the culture media was removed, 200 μl of culture media without FBS, and 20 μl of MTT (5 mg/ml in PBS) were added to each well, and the plate was returned to the incubator. After 4 hr, the media was removed from the wells, and the resulting formazan was solubilized with 200 μl of DMSO. Finally, the samples’ optical densities (ODs) were measured at 570 nm against 630 nm using an ELISA reader, and the percentage of cell survival was calculated compared to the control. Subsequently, the exposure time was determined.
Evaluation indicesThe time of CAP irradiation required a 50% decrease in cell survival (ET₅₀) and synergistic ratio (SYN). The synergistic ratio was defined (21) to compare two treatment groups with each other. The synergistic effect was calculated for each agent by quantifying the ratio between observed and anticipated cell death. It is expressed as follows:
The synergistic ratio of MB is the ratio of cell death (CD) caused by radiation in the presence of MB to the total cell death caused by radiation in the presence of each medicinal component individually.
$\mathit{Syn}\left(\mathit{MB}+\mathrm{CAP}\right)=\frac{\mathrm{CD}(\mathrm{MB}+\mathrm{CAP})}{\mathrm{CD}\left(\mathrm{MB}\right)+\mathrm{CD}\left(\mathrm{CAP}\right)}$ (2)
$\mathit{Syn}\left(\mathrm{Micelle}+\mathrm{CAP}\right)=\frac{\mathrm{CD}(\mathrm{Micelle}+\mathrm{CAP})}{\mathrm{CD}\left(\mathrm{Micelle}\right)+\mathrm{CD}\left(\mathrm{CAP}\right)}$ (3)